Individual body weights are determined by a complex blending of environmental and genetic influences. Population averages can demonstrate environmental influences while statistical genetics demonstrates biological influences on obesity. However, these methods cannot explain individual differences in body weight, i.e., why some people are obese and some are not. The individual causes for obesity may be determined by finding specific genes with natural variants (alleles) whose function varies among people. Many obesity genes have been identified, but others remain undiscovered. The principal investigator's (PI) goals are to identify obesity genes in vivo, to understand how they influence body fat accumulation, and to continue their in vitro work on the structure and function of uncoupling proteins (UCPs), which are candidate obesity genes. To study the basis for body weight regulation, the PI will: 1) determine the gene(s) that underlies obesity in a congenic mouse model, 2) study the mechanisms for uncoupling protein action, and 3) search for novel genes regulated by diet in congenic mouse strains. The PI and his collaborators have recently demonstrated that the C7.H1 congenic strain, which is significantly leaner than its background strain, has two linked chromosomal obesity loci, despite being 99 percent genetically identical to the background. They now propose to produce five subcongenic strains (with smaller donor regions) to identify the gene that underlies each locus. UCP2 and 3 and tubby are positional candidates for the distal locus. The PI will test their role by determining sequence from the congenic and background strains and by measurement of mRNA and protein levels. They will also use mRNA expression profiles from the C7.H1 congenic and subcongenic strains to identify novel obesity candidate genes. The PI will extend his studies of UCP2-3 as positional candidate genes for the C7.H1 congenic model by the application of structure/function studies of UCPs. Uncoupling proteins may influence body weight and metabolic rate by decreasing the efficacy with which mitochondria convert calories into ATP. Published data, and preliminary data in this proposal, demonstrate that UCPs are regulated by several ligands, such as retinoids, nucleotides, and fatty acids. The PI will test proton transport activity of established and novel candidate UCPs in liposomes and will study UCP structure by Site-Directed Spin Labeling (SDSL) Electron Paramagnetic Resonance (EPR). These studies will be coordinated with site-directed mutagenesis to test the hypotheses about mechanisms for proton transport.